AbstractThe C-terminal loop of the blue copper protein amicyanin, which contains three of the four active site ligands, has been replaced with a CuA binding loop. The purple protein produced has visible and EPR spectra identical to those of a CuA centre. Recent evidence strongly suggests that the CuA centre of cytochrome c oxidase and the A centre of nitrous oxide reductase are similar and are both binuclear. It therefore follows that the purple amicyanin mutant created here also possesses a binuclear CuA centre

Canters, Gerard W.

de Vries, Simon

Dennison, Christopher

van der Oost, John

Vijgenboom, Erik

Elsevier - Publisher Connector 

Introduction of a CuA site into the blue copper protein amicyanin from Thiobacillus versutus 

Dennison, C.

Vijgenboom, E.

Vries, S. de

Oost, J. van der

Canters, G.W.

Leiden University Scholary Publications

Introduction of a Cu_A site into the blue copper protein amicyanin fromthiobacillus-versutusDennison, C.; Vijgenboom, E.; Vries, S. de; Oost, J. van der; Canters, G.W.CitationDennison, C., Vijgenboom, E., Vries, S. de, Oost, J. van der, & Canters, G. W. (1995).Introduction of a Cu_A site into the blue copper protein amicyanin from thiobacillus-versutus.Febs Letters, 365(1), 92-94. doi:10.1016/0014-5793(95)00429-D Version: Publisher's VersionLicense: Licensed under Article 25fa Copyright Act/Law (Amendment Taverne)Downloaded from: https://hdl.handle.net/1887/3239418 Note: To cite this publication please use the final published version (if applicable).FEBS Letters 365 (1995) 92 94 FEBS 15501 Introduction of a CUA site into the blue copper protein amicyanin from Thiobacillus versutus Christopher Dennison a, Erik Vijgenboom a, Simon de Vries b, John van der Oost c, Gerard  W. Canters  a'* ~Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands bDepartment of Microbiology and Enzymology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, The Netherlands CDepartment of Molecular and Cellular Biology, BioCentrum Amsterdam, Vrije Universiteit, DeBoelelaan 1087, 1081 HV Amsterdam, The Netherlands Received 13 April 1995 Abstract The C-terminal loop of the blue copper protein amicy- anin, which contains three of the four active site ligands, has been replaced with a CUA binding loop. The purple protein produced has visible and EPR spectra identical to those of a CuA centre. Recent evidence strongly suggests that the CuA centre of cyto- chrome c oxidase and the A centre of nitrous oxide reductase are similar and are both binuclear. It therefore follows that the purple amicyanin mutant created here also possesses a binuclear Cu A centre. Key words: Copper protein; Site-directed mutagenesis; Amicyanin;  Blue copper 1. Introduction Cytochrome c oxidases are the terminal electron acceptors in aerobic respiratory chains and hence catalyse the reduction of dioxygen to water [1-5]. The aa3-type cytochrome c oxidases possess two functional subunits. Subunit I contains the binu- clear oxygen binding site which comprises a high-spin haem iron (cytochrome a3) and a copper site referred to as CuB. This subunit  also contains a low-spin haem site known as cyto- chrome a. Subunit  II contains a copper site [6] which, along with cytochrome a of subunit  I, is thought to be involved in the passage of electrons from the cytochrome c donor to the oxygen binding site. The copper site in subunit II is known as CUA and recent studies indicate that it is binuclear [6-13]. The suggestion that the Cu A centre of cytochrome c oxidase may be binuclear has existed for many years [14]. However, this proposal was usually met with a large degree of scepticism, and only recent studies on nitrous oxide reductase (N2OR) have led to the universal acceptance of this fact, EPR studies on N2OR indicated that its A centre contains two copper atoms in a mixed valence [Cu(1.5)...Cu(1.5)] site [15]. Similarities between the EPR spectrum of the A centre of N2OR and that of the CUA site of cytochrome c oxidase led to the conclusion that CUA is also binuclear [12,13]. The ligands to the two copper atoms in CUA are probably two cysteines, two histidines and a methion- ine [7]. Recent EXAFS studies on a soluble CUA domain of cytochrome c oxidase reveal that the two copper atoms are within 2.5 A of each other [16]. The authors of this work proposed a model for the CUA site in which there is a C u -C u  bond and in which the individual copper atoms are coordinated by a cysteine, a histidine and a third residue, which for one of *Corresponding author. Fax: (31) (71) 274 349. E-mail: CANTERS@Rulgca.LeidenUniv.nl the copper atoms is believed to be a methionine. An alternative binuclear model has recently been proposed [11] containing no bond between the copper atoms but in which the two cysteine residues act as bridging ligands. Resonance Raman studies, utilising the copper thiolate chromophore present in the CUA site ofcytochrome c oxidase and the A centre ofN2OR , demon- strate that only one Cu-S(Cys) stretching vibration is detectable for these two sites [17]. This is thought to be due to the two Cys ligands being spectroscopically equivalent which is claimed to be consistent with the EXAFS model. Type I blue copper proteins (cupredoxins) have a single cop- per atom at their active site which is usually coordinated by a cysteine, two histidines and a methionine, in a distorted tetrahe- dral arrangement [18]. Three of these ligands (the Cys, a His and the Met) are found close together in the C-terminal se- quence. From sequence alignments this loop in the cupredoxins corresponds to the CUA binding region of subunit  II of  cyto- chrome c oxidase (COII). The main difference between the type I copper and CUA binding proteins appears to be the length of this l igand-containing loop, with the latter having a longer loop possessing an extra cysteine residue (Table 1). Mutagenesis studies have shown that a type I blue copper site and a Cu~, site can be introduced into subunit  II of the o-quinol oxidase from Escherichia coli, which naturally lacks both of these sites [6], confirming that a cupredoxin-like domain is present in subunit II of both cytochrome c and o-quinol oxidases. In this article we show that a CUA site can be introduced into the blue copper protein amicyanin from Thiobacillus versutus. 2. Materials and methods 2.1. Construction of the Cu A mutation The sequence from Thr-94 to Phe-98 in wild-type amicyanin was replaced with the sequence Ala-Glu-Ile-Cys-Gly-Pro-Gly-His-Ser-Gly- (Table 1) using a modified version of the unique-site elimination mut- agenesis protocol [19]. The sequence introduced is identical to that which was used to produce a CuA binding mutant in the o-quinol oxidase (Table 1)[6]. This mutation, as well as providing the proposed ligands, introduces glutamate and isoleucine/leucine residues between the two cysteines, and a glycine following the second cysteine. All of these amino acids are conserved in COII sequences [2]. 2.2. Expression and purification E.coli BL21 [20] was transformed with a pUCI8 derivative harbour- ing the amicyanin construct containing the CuA mutation under the control of the lac promotor. The procedure used for expression and purification was a modified version of that described previously [21]. All of the pre-cultures contained Cu(NO3)2 to a concentration of 100 /2M. The cells were allowed to grow at 37°C for only 3 h after induction. Longer incubation times between induction and harvesting were found to result in a decrease in the amount of protein present in the cells. The 0014-5793/95l$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved. S S D I  0014-5793(95)00429-7 0.2 cells were resuspended in sucrose buffer (20% sucrose, 30 mM Tris and 1 mM EDTA, pH 8.0) and the periplasmic proteins were released by a single cycle of freezing and thawing. The final CM column, at pH 4.5, used in the wild-type procedure was replaced with purification on an FPLC Mono-Q column (Pharmacia) in 20 mM Tris buffer at pH 7.5. Elution of the protein was achieved using an ionic strength gradient created by 1 M NaCI. The C u  g domain of the caa3-type cytochrome c oxidase from Bacillus subtilis was isolated as described previously [9]. SDS-PAGE and Western blotting were carried out using a Bio-Rad mini protein II system. Western blotting was carried out according to Sambrook et al. [22] using Hybond C super membrane (Amersham) and polyclonal antibodies raised in rabbit against amicyanin. SDS-PAGE was also performed on a Pharmacia PhastSystem. 2.3. Spectroscopic characterisation For the spectroscopic measurements the Cu a amicyanin mutant was exchanged, using ultrafiltration (Amicon, YM5 membrane), into 50 mM HEPES buffer, pH 7.0. The visible spectrum was obtained on a Shimadzu UV-2101PC spectrophotometer at 25°C. The X-band EPR spectrum was obtained on a Varian E-9 spectrometer with a home- made cryostat operating at 23 K. For the EPR measurements the protein solution was mixed with an equal volume of 87% glycerol. 3. Results and discussion During the isolation procedure of  the CuA amicyanin mutant  a purple protein was observed which was obtained as a rela- tively pure fraction from the F P L C  Mono-Q column. This protein gave a main band on an SDS-PAGE gel with a slightly larger molecular weight than wild-type amicyanin. This band comprised >90% of  the sample and was detected with polyclo- nal antibodies raised against amicyanin. The CUA mutat ion in amicyanin introduces an extra cysteine into the primary structure of  the protein which could lead to the formation of  dimers via disulfide bridges. To investigate the possibility of  dimer formation the mutant  was analysed by SDS-PAGE in a similar way as to that used by Kumer  et al. [23] for Parococcus denitrificans amicyanin. A sample of  the CuA mutant  of  amicyanin which was pre-treated in soley SDS- PAGE buffer, prior to electrophoresis, was run on a gel along with a sample pretreated in SDS-PAGE buffer and heated prior to electrophoresis, and a sample pretreated in SDS-PAGE buffer plus/~-mercaptoethanol and heated prior to electropho- resis. In all cases the purple CUA amicyanin mutant  gave a band corrsponding to a monomer.  As a control, these experiments were repeated using wild-type Thiobacillus versutus amicyanin. This protein gave a monomer  except when the sample was heated prior to electrophoresis in the absence of  fl-mercap- toethanol,  when a dimer was observed. These results are iden- tical to those published for amicyanin from P. denitrificans [23]. The visible spectrum of  the purple CUA amicyanin mutant  is 0.15 0.1 0.05 i i 400 500 600 700 800 C Dennison et al./FEBS Letters 365 (1995) 92 94 93 a b i i i i 400 500 600 700 800 Wavelength (nm) Fig. 1. Visible spectrum of (a) the CuA-containing amicyanin mutant (25°C) in 50 mM HEPES buffer at pH 7.0 and (b) the CUA domain of cytochrome c oxidase from B. subtilis (as in [9]). shown in Fig. la  and has peaks at 360, 483, 532 and a broad absorption at approximately 790 nm. The spectrum is almost identical to that of  the native CUA domain of  cytochrome c oxidase from B. subtilis (Fig. lb) and is similar to those of  the native CUA domain of  cytochrome c oxidase from Paracoccus denitrificans [8], the Cu A binding mutant  of  the o-quinol oxidase [6] and the A centre of  N2OR [17]. Resonance Raman studies on the CUA domain of  cytochrome c oxidase from B. subtilis have shown that excitation of  the three visible absorption bands above 400 nm produces the same set of  R R  frequencies. These bands are therefore assigned to different electronic transitions of  the same Cu-S(Cys) chromophore.  Table 1 Partial amino acid sequence of Thiobacillus versutus amicyanin showing the C-terminal loop which contains three of the four active site ligands Protein Amino acid sequence Amicyanin Cys Thr Pro His Pro Phe Met 99 COI! Cys Ala Glu Leu Cys Gly Pro Ser His Ala Leu Met 227 N2OR Cys Ser Trp Phe Cys His Ala Leu His Met Glu Met 628 CyoA Ser Ala Ser Tyr Ser Gly Pro Gly Phe Ser Gly Met 219 CyoA* Cys Ala Glu Ile Cys Gly Pro Gly His Ser Gly Met 219 Also shown are the homologous sequences from subunit II of cytochrome c oxidase from Bacillus subtilis (COII), nitrous oxide reductase from Pseudomonas stutzeri (N2OR), cytochrome o-quinol oxidase from Escherichia coli (CyoA) and the CUA binding mutant ofcytochrome o-quinol oxidase (CyoA*) [6]. In all cases the (proposed) copper ligands are in bold and the position of the final residue (methionine) shown, in the primary structure of the respective proteins, is indicated in the final column. 94 C. Dennison et al./FEBS Letters 365 (1995) 92-94 a I I [ I I 260 280 a00 a20 340 magnetic field (mT) Fig. 2. X-band EPR spectrum at 23 K and using a power level at which the type II signal is saturated (20 mW) of(a) the CUA-containing amicy- anin mutant in 25 mM HEPES (~40% glycerol) at pH 7.0 and (b) the Cu a domain of cytochrome c oxidase from B. subtilis in 20 mM Tris buffer (10% glycerol) at pH 8.0. The EPR spectrum of the Cu A amicyanin mutant  contains signals from two Cu(II) species which are of  approximately equal intensity. One is a distinctive type II copper site which becomes saturated at high power levels. Similar signals are also found for the CuA-containing mutant  of  the soluble fragment of  subunit II of  cytochrome o-quinol oxidase [6], the soluble CUA domain of  cytochrome c oxidase from B. subtilis [9] and also in another active site mutant  od amicyanin [24]. In all cases this is assigned to adventitiously bound copper. The second EPR signal is characteristic of  a Cu A centre and is shown in Fig. 2 along with the spectrum of  the CuA-contain- ing domain of  cytochrome c oxidase from B. subtilis. The EPR parameters for these two spectra are shown in Table 2. The spectrum of  the CUA amicyanin mutant  is also similar to other published Cu A EPR spectra [6,8]. The 7-line hyperfine struc- ture, which is consistent with one unpaired electron interacting with two S = 3/2 nuclei, i.e. in a mixed valence binuclear site, is only fully resolved at X-band frequency for the A centre of  N2OR, but not completely for any of  the CUA-Containing pro- teins. The fine structure of  the Gz signal in the case of  the CUA mutant  of  amicyanin is consistent with a 7-line pattern which is more resolved than in the B. subtilis CUA spectrum, despite the smaller A z value in the former. Table 2 EPR parameters for the CuA amicyanin mutant (CuAami) and the Cun domain of cytochrome c oxidase from B. subtilis (B2) Protein gz Az (roT) gx,y CuAami 2.18 3.24 1.99 2.02 B2 2.18 3.82 1.99 2.03 In conclusion, the purple amicyanin presented here seems to bind copper as a CUA site. Since this site has been shown to be binuclear in the soluble CUA domains of  cytochrome c oxidase and also in the CUA-containing mutant  of  the o-quinol oxidase it follows that the site created in amicyanin also binds two copper atoms. This work confirms the idea that the CUA do- main of  the oxidases possesses a cupredoxin-like fold. Work is currently underway to further characterise this amicyanin mu- tant. Acknowledgements: This research was funded, in part, by the EC Sci- ence project SCI-CT90-0434 along with the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organisation for Scientific Research (NWO), and under the auspices of the BIOMAC Graduate Research School of Leiden and Delft. References [1] Malmstr6m, B.G. (1990) Chem. Rev. 90, 1247-1260. [2] Saraste, M. (1990) Quat Rev. Biophys. 23, 331 366. [3] Babcock, G.T. and Wikstr6m, M. (1992) Nature 356, 301 309. [4] Calhoun, M.W., Thomas, J.W. and Gennis, R.B. (1994) Trends Biochem. Sci. 19, 325 330. [5] Garcia-Horsman, J.A., Barquera, B., Rumbley, J., Ma, J. and Gennis, R.B. (1994) J. Bacteriol. 176, 5587-5600. [6] van der Oost, J., Lappalainen, P., Musacchio, A., Warne, A., Lemieux, L., Rumbley, J., Gennis, R.B., Aasa, R., Pascher, T., Malmstr6m, B.G. and Saraste, M. (1992) EMBO J. 11,3209-3217. [7] Kelly, M., Lappalainen, P., Talbo, G., Haltia, T., van der Oost, J. and Saraste, M. (1993) J. Biol. Chem. 268, 16781-16787. [8] Lappalainen, P., Aasa, R., Malmstr6m, B.G. and Saraste, M. (1993) J. Biol. Chem. 268, 26416-26421. [9] von Wachenfeldt, C., de Vries, S. and van der Oost, J. (1994) FEBS Lett. 340, 109-113. [10] Malmstr6m, B.G. and Aasa, R. (1993) FEBS Lett. 325, 49-52. [11] Lappalainen, E and Saraste, M. (1994) Biochim. Biophys. Acta 1187, 222-225. [12] Kroneck, P.M.H., Antholine, W.A., Kastrau, D.H.W., Buse, G., Steffens, G.C.M. and Zumft, W.G. (1990) FEBS Lett. 268, 274- 276. [13] Antholine, W.E., Kastrau, D.H.W., Steffens, G.C.M., Buse, G., Zumft, W.G. and Kroneck, P.M.H. (1992) Eur. J. Biochem. 209, 875--881. [14] Beinert, H., Griffiths, D.E., Wharton, D.C. and Sands, R.H. (1962) J. Biol. Chem. 237, 2337-2346. [15] Kroneck, P.M.H., Antholine, W.A., Reister, J. and Zumft, W.G. (1988) FEBS Lett. 242, 70-74. [16] Blackburn, N.J., Barr, M.E., Woodruff; W.H., van der Oost, J. and de Vries, S. (1994) Biochemistry 33, 10401 10407. [17] Andrew, C.R., Han, J., de Vries, S., van der Oost, J., Averill, B.A., Loehr, T.M. and Sanders-Loehr, J. (1994) J. Am. Chem. Soc. 116, 10805-10806. [18] Adman, E.T. (1991) Curr. Opin. Struct. Biol. l, 895-904. [19] Deng, W.E and Nickoloff, J.A. (1992) Anal. Biochem. 200, 81 88. [20] Studier, F.W. and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113 130. [21] Kalverda, A.E, Wymenga, S.S., Lommen, A., van de Ven, F.J.M., Hilbers, C.W. and Canters, G.W. (1994) J. Mol. Biol. 240, 358 371. [22] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn., CSH Laboratory Press, NY. [23] Kumer, M.A. and Davidson, V.L. (1992) BioTechniques 12, 198- 201. [24] Dennison, C. and Canters, G.W., unpublished data. 

Introduction of a Cu_A site into the blue copper protein amicyanin from thiobacillus-versutus

FEBS Letters 365 (1995) 92 94 FEBS 15501 
Introduction of a CUA site into the blue copper protein amicyanin from 
Thiobacillus versutus 
Christopher Dennison a, Erik Vijgenboom a, Simon de Vries b, John van der Oost c, 
Gerard W. Canters a'* 
~Leiden Institute of Chemistry, Gorlaeus Laboratories, Einsteinweg 55, PO Box 9502, 2300 RA Leiden, The Netherlands 
bDepartment of Microbiology and Enzymology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, The Netherlands 
CDepartment ofMolecular and Cellular Biology, BioCentrum Amsterdam, Vrije Universiteit, DeBoelelaan 1087, 1081 HV Amsterdam, The Netherlands 
Received 13 April 1995 
Abstract The C-terminal oop of the blue copper protein amicy- 
anin, which contains three of the four active site ligands, has been 
replaced with a CUA binding loop. The purple protein produced 
has visible and EPR spectra identical to those of a CuA centre. 
Recent evidence strongly suggests that the CuA centre of cyto- 
chrome c oxidase and the A centre of nitrous oxide reductase are 
similar and are both binuclear. It therefore follows that the purple 
amicyanin mutant created here also possesses a binuclear Cu A 
centre. 
Key words: Copper protein; Site-directed mutagenesis; 
Amicyanin; Blue copper 
1. Introduction 
Cytochrome c oxidases are the terminal electron acceptors 
in aerobic respiratory chains and hence catalyse the reduction 
of dioxygen to water [1-5]. The aa3-type cytochrome c oxidases 
possess two functional subunits. Subunit I contains the binu- 
clear oxygen binding site which comprises a high-spin haem 
iron (cytochrome a3) and a copper site referred to as CuB. This 
subunit also contains a low-spin haem site known as cyto- 
chrome a. Subunit II contains a copper site [6] which, along 
with cytochrome a of subunit I, is thought o be involved in the 
passage of electrons from the cytochrome c donor to the oxygen 
binding site. The copper site in subunit II is known as CUA and 
recent studies indicate that it is binuclear [6-13]. 
The suggestion that the Cu A centre of cytochrome c oxidase 
may be binuclear has existed for many years [14]. However, this 
proposal was usually met with a large degree of scepticism, and 
only recent studies on nitrous oxide reductase (N2OR) have led 
to the universal acceptance of this fact, EPR studies on N2OR 
indicated that its A centre contains two copper atoms in a 
mixed valence [Cu(1.5)...Cu(1.5)] site [15]. Similarities between 
the EPR spectrum of the A centre of N2OR and that of the CUA 
site of cytochrome c oxidase led to the conclusion that CUA is 
also binuclear [12,13]. The ligands to the two copper atoms in 
CUA are probably two cysteines, two histidines and a methion- 
ine [7]. Recent EXAFS studies on a soluble CUA domain of 
cytochrome c oxidase reveal that the two copper atoms are 
within 2.5 A of each other [16]. The authors of this work 
proposed a model for the CUA site in which there is a Cu-Cu 
bond and in which the individual copper atoms are coordinated 
by a cysteine, a histidine and a third residue, which for one of 
*Corresponding author. Fax: (31) (71) 274 349. 
E-mail: CANTERS@Rulgca.LeidenUniv.nl 
the copper atoms is believed to be a methionine. An alternative 
binuclear model has recently been proposed [11] containing no 
bond between the copper atoms but in which the two cysteine 
residues act as bridging ligands. Resonance Raman studies, 
utilising the copper thiolate chromophore present in the CUA 
site ofcytochrome c oxidase and the A centre ofN2OR , demon- 
strate that only one Cu-S(Cys) stretching vibration is detectable 
for these two sites [17]. This is thought o be due to the two Cys 
ligands being spectroscopically equivalent which is claimed to 
be consistent with the EXAFS model. 
Type I blue copper proteins (cupredoxins) have a single cop- 
per atom at their active site which is usually coordinated by a 
cysteine, two histidines and a methionine, ina distorted tetrahe- 
dral arrangement [18]. Three of these ligands (the Cys, a His 
and the Met) are found close together in the C-terminal se- 
quence. From sequence alignments his loop in the cupredoxins 
corresponds to the CUA binding region of subunit II of cyto- 
chrome c oxidase (COII). The main difference between the type 
I copper and CUA binding proteins appears to be the length of 
this ligand-containing loop, with the latter having a longer loop 
possessing an extra cysteine residue (Table 1). Mutagenesis 
studies have shown that a type I blue copper site and a Cu~, site 
can be introduced into subunit II of the o-quinol oxidase from 
Escherichia coli, which naturally lacks both of these sites [6], 
confirming that a cupredoxin-like domain is present in subunit 
II of both cytochrome c and o-quinol oxidases. In this article 
we show that a CUA site can be introduced into the blue copper 
protein amicyanin from Thiobacillus versutus. 
2. Materials and methods 
2.1. Construction of the Cu A mutation 
The sequence from Thr-94 to Phe-98 in wild-type amicyanin was 
replaced with the sequence Ala-Glu-Ile-Cys-Gly-Pro-Gly-His-Ser-Gly- 
(Table 1) using a modified version of the unique-site elimination mut- 
agenesis protocol [19]. The sequence introduced is identical to that 
which was used to produce a CuA binding mutant in the o-quinol 
oxidase (Table 1)[6]. This mutation, as well as providing the proposed 
ligands, introduces glutamate and isoleucine/leucine residues between 
the two cysteines, and a glycine following the second cysteine. All of 
these amino acids are conserved in COII sequences [2]. 
2.2. Expression and purification 
E.coli BL21 [20] was transformed with a pUCI8 derivative harbour- 
ing the amicyanin construct containing the CuA mutation under the 
control of the lac promotor. The procedure used for expression and 
purification was a modified version of that described previously [21]. 
All of the pre-cultures contained Cu(NO3)2 to a concentration f 100 
/2M. The cells were allowed to grow at 37°C for only 3 h after induction. 
Longer incubation times between induction and harvesting were found 
to result in a decrease in the amount of protein present in the cells. The 
0014-5793/95l$9.50 © 1995 Federation of European Biochemical Societies. All rights reserved. 
SSDI  0014-5793(95)00429-7 
0.2 cells were resuspended in sucrose buffer (20% sucrose, 30 mM Tris and 
1 mM EDTA, pH 8.0) and the periplasmic proteins were released by 
a single cycle of freezing and thawing. The final CM column, at pH 4.5, 
used in the wild-type procedure was replaced with purification on an 
FPLC Mono-Q column (Pharmacia) in 20 mM Tris buffer at pH 7.5. 
Elution of the protein was achieved using an ionic strength gradient 
created by 1 M NaCI. 
The Cu g domain of the caa3-type cytochrome c oxidase from Bacillus 
subtilis was isolated as described previously [9]. 
SDS-PAGE and Western blotting were carried out using a Bio-Rad 
mini protein II system. Western blotting was carried out according to 
Sambrook et al. [22] using Hybond C super membrane (Amersham) and 
polyclonal antibodies raised in rabbit against amicyanin. SDS-PAGE 
was also performed on a Pharmacia PhastSystem. 
2.3. Spectroscopic characterisation 
For the spectroscopic measurements the Cu a amicyanin mutant was 
exchanged, using ultrafiltration (Amicon, YM5 membrane), into 50 
mM HEPES buffer, pH 7.0. The visible spectrum was obtained on a 
Shimadzu UV-2101PC spectrophotometer at 25°C. The X-band EPR 
spectrum was obtained on a Varian E-9 spectrometer with a home- 
made cryostat operating at 23 K. For the EPR measurements the 
protein solution was mixed with an equal volume of 87% glycerol. 
3. Results and discussion 
During the isolation procedure of the CuA amicyanin mutant 
a purple protein was observed which was obtained as a rela- 
tively pure fraction from the FPLC Mono-Q column. This 
protein gave a main band on an SDS-PAGE gel with a slightly 
larger molecular weight than wild-type amicyanin. This band 
comprised >90% of the sample and was detected with polyclo- 
nal antibodies raised against amicyanin. 
The CUA mutation in amicyanin introduces an extra cysteine 
into the primary structure of the protein which could lead to 
the formation of dimers via disulfide bridges. To investigate the 
possibility of dimer formation the mutant was analysed by 
SDS-PAGE in a similar way as to that used by Kumer et al. 
[23] for Parococcus denitrificans amicyanin. A sample of the 
CuA mutant of amicyanin which was pre-treated in soley SDS- 
PAGE buffer, prior to electrophoresis, was run on a gel along 
with a sample pretreated in SDS-PAGE buffer and heated prior 
to electrophoresis, and a sample pretreated in SDS-PAGE 
buffer plus/~-mercaptoethanol andheated prior to electropho- 
resis. In all cases the purple CUA amicyanin mutant gave a band 
corrsponding to a monomer. As a control, these experiments 
were repeated using wild-type Thiobacillus versutus amicyanin. 
This protein gave a monomer except when the sample was 
heated prior to electrophoresis n the absence of fl-mercap- 
toethanol, when a dimer was observed. These results are iden- 
tical to those published for amicyanin from P. denitrificans [23]. 
The visible spectrum of the purple CUA amicyanin mutant is 
0.15 
0.1 
0.05 i i 
400 500 600 700 800 
C Dennison et al./FEBS Letters 365 (1995) 92 94 93 
a 
b 
i i i i 
400 500 600 700 800 
Wavelength (nm) 
Fig. 1. Visible spectrum of (a) the CuA-containing amicyanin mutant 
(25°C) in 50 mM HEPES buffer at pH 7.0 and (b) the CUA domain of 
cytochrome c oxidase from B. subtilis (as in [9]). 
shown in Fig. la and has peaks at 360, 483, 532 and a broad 
absorption at approximately 790 nm. The spectrum is almost 
identical to that of the native CUA domain of cytochrome c
oxidase from B. subtilis (Fig. lb) and is similar to those of the 
native CUA domain of cytochrome c oxidase from Paracoccus 
denitrificans [8], the Cu A binding mutant of the o-quinol oxidase 
[6] and the A centre of N2OR [17]. Resonance Raman studies 
on the CUA domain of cytochrome c oxidase from B. subtilis 
have shown that excitation of the three visible absorption bands 
above 400 nm produces the same set of RR frequencies. These 
bands are therefore assigned to different electronic transitions 
of the same Cu-S(Cys) chromophore. 
Table 1 
Partial amino acid sequence of Thiobacillus versutus amicyanin showing the C-terminal loop which contains three of the four active site ligands 
Protein Amino acid sequence 
Amicyanin Cys Thr Pro His Pro Phe Met 99 
COI! Cys Ala Glu Leu Cys Gly Pro Ser His Ala Leu Met 227 
N2OR Cys Ser Trp Phe Cys His Ala Leu His Met Glu Met 628 
CyoA Ser Ala Ser Tyr Ser Gly Pro Gly Phe Ser Gly Met 219 
CyoA* Cys Ala Glu Ile Cys Gly Pro Gly His Ser Gly Met 219 
Also shown are the homologous sequences from subunit II of cytochrome c oxidase from Bacillus subtilis (COII), nitrous oxide reductase from 
Pseudomonas stutzeri (N2OR), cytochrome o-quinol oxidase from Escherichia coli (CyoA) and the CUA binding mutant ofcytochrome o-quinol oxidase 
(CyoA*) [6]. In all cases the (proposed) copper ligands are in bold and the position of the final residue (methionine) shown, in the primary structure 
of the respective proteins, is indicated in the final column. 
94 C. Dennison et al./FEBS Letters 365 (1995) 92-94 
a 
I I [ I I 
260 280 a00 a20 340 
magnetic field (mT) 
Fig. 2. X-band EPR spectrum at 23 K and using a power level at which 
the type II signal is saturated (20 mW) of(a) the CUA-containing amicy- 
anin mutant in 25 mM HEPES (~40% glycerol) at pH 7.0 and (b) the 
Cu a domain of cytochrome c oxidase from B. subtilis in 20 mM Tris 
buffer (10% glycerol) at pH 8.0. 
The EPR spectrum of the Cu A amicyanin mutant contains 
signals from two Cu(II) species which are of approximately 
equal intensity. One is a distinctive type II copper site which 
becomes aturated at high power levels. Similar signals are also 
found for the CuA-containing mutant of the soluble fragment 
of subunit II of cytochrome o-quinol oxidase [6], the soluble 
CUA domain of cytochrome c oxidase from B. subtilis [9] and 
also in another active site mutant od amicyanin [24]. In all cases 
this is assigned to adventitiously bound copper. 
The second EPR signal is characteristic of a Cu A centre and 
is shown in Fig. 2 along with the spectrum of the CuA-contain- 
ing domain of cytochrome c oxidase from B. subtilis. The EPR 
parameters for these two spectra are shown in Table 2. The 
spectrum of the CUA amicyanin mutant is also similar to other 
published Cu A EPR spectra [6,8]. The 7-line hyperfine struc- 
ture, which is consistent with one unpaired electron interacting 
with two S = 3/2 nuclei, i.e. in a mixed valence binuclear site, 
is only fully resolved at X-band frequency for the A centre of 
N2OR, but not completely for any of the CUA-Containing pro- 
teins. The fine structure of the Gz signal in the case of the CUA 
mutant of amicyanin is consistent with a 7-line pattern which 
is more resolved than in the B. subtilis CUA spectrum, despite 
the smaller A z value in the former. 
Table 2 
EPR parameters for the CuA amicyanin mutant (CuAami) and the Cun 
domain of cytochrome c oxidase from B. subtilis (B2) 
Protein gz Az (roT) gx,y 
CuAami 2.18 3.24 1.99 2.02 
B2 2.18 3.82 1.99 2.03 
In conclusion, the purple amicyanin presented here seems to 
bind copper as a CUA site. Since this site has been shown to be 
binuclear in the soluble CUA domains of cytochrome c oxidase 
and also in the CUA-containing mutant of the o-quinol oxidase 
it follows that the site created in amicyanin also binds two 
copper atoms. This work confirms the idea that the CUA do- 
main of the oxidases possesses a cupredoxin-like fold. Work is 
currently underway to further characterise this amicyanin mu- 
tant. 
Acknowledgements: This research was funded, in part, by the EC Sci- 
ence project SCI-CT90-0434 along with the Netherlands Foundation 
for Chemical Research (SON) with financial aid from the Netherlands 
Organisation for Scientific Research (NWO), and under the auspices 
of the BIOMAC Graduate Research School of Leiden and Delft. 
References 
[1] Malmstr6m, B.G. (1990) Chem. Rev. 90, 1247-1260. 
[2] Saraste, M. (1990) Quat Rev. Biophys. 23, 331 366. 
[3] Babcock, G.T. and Wikstr6m, M. (1992) Nature 356, 301 309. 
[4] Calhoun, M.W., Thomas, J.W. and Gennis, R.B. (1994) Trends 
Biochem. Sci. 19, 325 330. 
[5] Garcia-Horsman, J.A., Barquera, B., Rumbley, J., Ma, J. and 
Gennis, R.B. (1994) J. Bacteriol. 176, 5587-5600. 
[6] van der Oost, J., Lappalainen, P., Musacchio, A., Warne, A., 
Lemieux, L., Rumbley, J., Gennis, R.B., Aasa, R., Pascher, T., 
Malmstr6m, B.G. and Saraste, M. (1992) EMBO J. 11,3209-3217. 
[7] Kelly, M., Lappalainen, P., Talbo, G., Haltia, T., van der Oost, 
J. and Saraste, M. (1993) J. Biol. Chem. 268, 16781-16787. 
[8] Lappalainen, P., Aasa, R., Malmstr6m, B.G. and Saraste, M. 
(1993) J. Biol. Chem. 268, 26416-26421. 
[9] von Wachenfeldt, C., de Vries, S. and van der Oost, J. (1994) FEBS 
Lett. 340, 109-113. 
[10] Malmstr6m, B.G. and Aasa, R. (1993) FEBS Lett. 325, 49-52. 
[11] Lappalainen, E and Saraste, M. (1994) Biochim. Biophys. Acta 
1187, 222-225. 
[12] Kroneck, P.M.H., Antholine, W.A., Kastrau, D.H.W., Buse, G., 
Steffens, G.C.M. and Zumft, W.G. (1990) FEBS Lett. 268, 274- 
276. 
[13] Antholine, W.E., Kastrau, D.H.W., Steffens, G.C.M., Buse, G., 
Zumft, W.G. and Kroneck, P.M.H. (1992) Eur. J. Biochem. 209, 
875--881. 
[14] Beinert, H., Griffiths, D.E., Wharton, D.C. and Sands, R.H. 
(1962) J. Biol. Chem. 237, 2337-2346. 
[15] Kroneck, P.M.H., Antholine, W.A., Reister, J. and Zumft, W.G. 
(1988) FEBS Lett. 242, 70-74. 
[16] Blackburn, N.J., Barr, M.E., Woodruff; W.H., van der Oost, J. 
and de Vries, S. (1994) Biochemistry 33, 10401 10407. 
[17] Andrew, C.R., Han, J., de Vries, S., van der Oost, J., Averill, B.A., 
Loehr, T.M. and Sanders-Loehr, J. (1994) J. Am. Chem. Soc. 116, 
10805-10806. 
[18] Adman, E.T. (1991) Curr. Opin. Struct. Biol. l, 895-904. 
[19] Deng, W.E and Nickoloff, J.A. (1992) Anal. Biochem. 200, 81 
88. 
[20] Studier, F.W. and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113 
130. 
[21] Kalverda, A.E, Wymenga, S.S., Lommen, A., van de Ven, F.J.M., 
Hilbers, C.W. and Canters, G.W. (1994) J. Mol. Biol. 240, 358 
371. 
[22] Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular 
Cloning: A Laboratory Manual, 2nd edn., CSH Laboratory Press, 
NY. 
[23] Kumer, M.A. and Davidson, V.L. (1992) BioTechniques 12, 198- 
201. 
[24] Dennison, C. and Canters, G.W., unpublished ata. 


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Introduction of a CuA site into the blue copper protein amicyanin from Thiobacillus versutus

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